Ryu Ae Jin, Shin Won-Sub, Jang Sunghoon, Lin Yejin, Park Yejee, Choi Yujung, Kim Ji Young, Kang Nam Kyu
CJ BIO Research Institute , CJ CheilJedang, Suwon-si, Gyeonggi- do, 16495, Republic of Korea.
Department of Chemical Engineering, College of Engineering, Kyung Hee University, Yongin, 17104, Republic of Korea.
J Biol Eng. 2024 Oct 10;18(1):56. doi: 10.1186/s13036-024-00447-y.
Schizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production.
Transgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain.
This study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.
裂殖壶菌是一类真核海洋原生生物，是产油菌株，这使其成为生产生物燃料和omega-3脂肪酸等脂质衍生产品的极具潜力的候选者。然而，基因工程工具发展不足阻碍了进一步的进展。因此，开发用于提高脂质产量的基因工程工具并将其应用于工业生产至关重要。
裂殖壶菌中的转基因表达常常面临挑战，例如由于位置效应导致的不稳定性。为克服这一问题，我们开发了一种安全港转基因表达系统。最初，将sfGFP基因随机整合，并使用荧光激活细胞分选鉴定高表达转化体。值得注意的是，位于细胞色素c上游约3.2 kb处的HRsite 2显示出增强的sfGFP表达和同源重组效率。然后我们在HRsite 2处引入3-酮脂酰-ACP还原酶（KR）基因，从而提高了脂质和二十二碳六烯酸（DHA）的产量。与随机整合转基因的转化体相比，在HRsite 2处带有KR的转化体表现出稳定的生长、更高的葡萄糖利用率和更高的脂质含量。值得注意的是，这些转化体的DHA含量比野生型菌株提高了25%。
本研究通过鉴定可靠的基因整合安全港位点，成功在裂殖壶菌中建立了强大的同源重组系统。KR基因在该位点的靶向表达不仅提高了DHA产量，还维持了生长和葡萄糖消耗率，验证了安全港方法的有效性。合成生物学和代谢工程的这一进展为裂殖壶菌中更高效的生物技术应用铺平了道路。
